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ItemNo Preview AvailableMof (MYST1 or KAT8) is essential for progression of embryonic development past the blastocyst stage and required for normal chromatin architecture(AMER SOC MICROBIOLOGY, 2008-08)Acetylation of histone tails is a hallmark of transcriptionally active chromatin. Mof (males absent on the first; also called MYST1 or KAT8) is a member of the MYST family of histone acetyltransferases and was originally discovered as an essential component of the X chromosome dosage compensation system in Drosophila. In order to examine the role of Mof in mammals in vivo, we generated mice carrying a null mutation of the Mof gene. All Mof-deficient embryos fail to develop beyond the expanded blastocyst stage and die at implantation in vivo. Mof-deficient cell lines cannot be derived from Mof(-/-) embryos in vitro. Mof(-/-) embryos fail to acetylate histone 4 lysine 16 (H4K16) but have normal acetylation of other N-terminal histone lysine residues. Mof(-/-) cell nuclei exhibit abnormal chromatin aggregation preceding activation of caspase 3 and DNA fragmentation. We conclude that Mof is functionally nonredundant with the closely related MYST histone acetyltransferase Tip60. Our results show that Mof performs a different role in mammals from that in flies at the organism level, although the molecular function is conserved. We demonstrate that Mof is required specifically for the maintenance of H4K16 acetylation and normal chromatin architecture of all cells of early male and female embryos.
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ItemOsteoclast Inhibitory Lectin, an Immune Cell Product That Is Required for Normal Bone Physiology in Vivo(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2008-11-07)Osteoclast inhibitory lectin (OCIL or clrb) is a member of the natural killer cell C-type lectins that have a described role mostly in autoimmune cell function. OCIL was originally identified as an osteoblast-derived inhibitor of osteoclast formation in vitro. To determine the physiological function(s) of OCIL, we generated ocil(-/-) mice. These mice appeared healthy and were fertile, with no apparent immune function defect, and phenotypic abnormalities were limited to bone. Histomorphometric analysis revealed a significantly lower tibial trabecular bone volume and trabecular number in the 10- and 16-week-old male ocil(-/-) mice compared with wild type mice. Furthermore, ocil(-/-) mice showed reduced bone formation rate in the 10-week-old females and 16-week-old males while Static markers of bone formation showed no significant changes in male or female ocil(-/-) mice. Examination of bone resorption markers in the long bones of ocil(-/-) mice indicated a transient increase in osteoclast number per unit bone perimeter. Enhanced osteoclast formation was also observed when either bone marrow or splenic cultures were generated in vitro from ocil(-/-) mice relative to wild type control cultures. Loss of ocil therefore resulted in osteopenia in adult mice primarily as a result of increased osteoclast formation and/or decreased bone formation. The enhanced osteoclastic activity led to elevated serum calcium levels, which resulted in the suppression of circulating parathyroid hormone in 10-week-old ocil(-/-) mice compared with wild type control mice. Collectively, our data suggest that OCIL is a physiological negative regulator of bone.
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ItemMacronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote.(Public Library of Science (PLoS), 2006-09)The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.
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ItemNo Preview AvailableFaces of integration.(Ubiquity Press, Ltd., 2009-12-22)THEME: Two central themes permeate this paper-the interplay between structure and agency in integration processes and the extent to which this is mediated through sensemaking by individual actors. CASE STUDY: The empirical base for the paper is provided by case study research from Wales which draws on examples of different types of integration in health and social care. The individual case studies highlight different interpretations of integration set against a background of the resources involved, processes employed and outcomes achieved. DISCUSSION: A wide ranging discussion exposes the complex interplay and dynamics between structural factors and the manner in which they enable or constrain integration, and individual actors realising their potential agency through leadership, professionalism and boundary spanning to influence outcomes. The importance of structure and agency complementing each other to determine effective integration is emphasised, together with the scope that is available for interpretation and meaning by individual actors within the contested discourse of integration.
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ItemNo Preview AvailableMembrane-bound Fas ligand only is essential for Fas-induced apoptosis(NATURE PUBLISHING GROUP, 2009-10-01)Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family, and its receptor Fas are critical for the shutdown of chronic immune responses and prevention of autoimmunity. Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice and humans. FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding. Here we generated gene-targeted mice that selectively lack either secreted FasL (sFasL) or membrane-bound FasL (mFasL) to resolve which of these forms is required for cell killing and to explore their hypothesized non-apoptotic activities. Mice lacking sFasL (FasL(Deltas/Deltas)) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL (FasL(Deltam/Deltam)) could not kill cells through Fas activation. FasL(Deltam/Deltam) mice developed lymphadenopathy and hyper-gammaglobulinaemia, similar to FasL(gld/gld) mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, FasL(Deltam/Deltam) mice (on a C57BL/6 background) succumbed to systemic lupus erythematosus (SLE)-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and much later in FasL(gld/gld) mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer, whereas excess sFasL appears to promote autoimmunity and tumorigenesis through non-apoptotic activities.
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ItemIn vitro sensitivity testing of minimally passaged and uncultured gliomas with TRAIL and/or chemotherapy drugs(NATURE PUBLISHING GROUP, 2008-07-15)TRAIL/Apo-2L has shown promise as an anti-glioma drug, based on investigations of TRAIL sensitivity in established glioma cell lines, but it is not known how accurately TRAIL signalling pathways of glioma cells in vivo are reproduced in these cell lines in vitro. To replicate as closely as possible the in vivo behaviour of malignant glioma cells, 17 early passage glioma cell lines and 5 freshly resected gliomas were exposed to TRAIL-based agents and/or chemotherapeutic drugs. Normal human hepatocytes and astrocytes and established glioma cell lines were also tested. Cross-linked TRAIL, but not soluble TRAIL, killed both normal cell types and cells from three tumours. Cells from only one glioma were killed by soluble TRAIL, although only inefficiently. High concentrations of cisplatin were lethal to glioma cells, hepatocytes and astrocytes. Isolated combinations of TRAIL and chemotherapy drugs were more toxic to particular gliomas than normal cells, but no combination was generally selective for glioma cells. This study highlights the widespread resistance of glioma cells to TRAIL-based agents, but suggests that a minority of high-grade glioma patients may benefit from particular combinations of TRAIL and chemotherapy drugs. In vitro sensitivity assays may help identify effective drug combinations for individual glioma patients.
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ItemUnderstanding the Relationship between Activity and Neighbourhoods (URBAN) Study: research design and methodology(BMC, 2009-07-10)BACKGROUND: Built environment attributes are recognized as being important contributors to physical activity (PA) engagement and body size in adults and children. However, much of the existing research in this emergent public health field is hindered by methodological limitations, including: population and site homogeneity, reliance on self-report measures, aggregated measures of PA, and inadequate statistical modeling. As an integral component of multi-country collaborative research, the Understanding the Relationship between Activity and Neighbourhoods (URBAN) Study seeks to overcome these limitations by determining the strengths of association between detailed measures of the neighborhood built environment with PA levels across multiple domains and body size measures in adults and children. This article outlines the research protocol developed for the URBAN Study. METHODS AND DESIGN: The URBAN Study is a multi-centered, stratified, cross-sectional research design, collecting data across four New Zealand cities. Within each city, 12 neighborhoods were identified and selected for investigation based on higher or lower walkability and Māori demographic attributes. Neighborhoods were selected to ensure equal representation of these characteristics. Within each selected neighborhood, 42 households are being randomly selected and an adult and child (where possible) recruited into the study. Data collection includes: objective and self-reported PA engagement, neighborhood perceptions, demographics, and body size measures. The study was designed to recruit approximately 2,000 adults and 250 children into the project. Other aspects of the study include photovoice, which is a qualitative assessment of built environment features associated with PA engagement, an audit of the neighborhood streetscape environment, and an individualized neighborhood walkability profile centered on each participant's residential address. Multilevel modeling will be used to examine the individual-level and neighborhood-level relationships with PA engagement and body size. DISCUSSION: The URBAN Study is applying a novel scientifically robust research design to provide urgently needed epidemiological information regarding the associations between the built environment and health outcomes. The findings will contribute to a larger, international initiative in which similar neighborhood selection and PA measurement procedures are utilized across eight countries. Accordingly, this study directly addresses the international priority issues of increasing PA engagement and decreasing obesity levels.
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ItemAustralian health policy on access to medical care for refugees and asylum seekers.(Springer Science and Business Media LLC, 2005-10-09)Since the tightening of Australian policy for protection visa applicants began in the 1990s, access to health care has been increasingly restricted to asylum seekers on a range of different visa types. This paper summarises those legislative changes and discusses their implications for health policy relating to refugees and asylum seekers in Australia. Of particular concern are asylum seekers on Bridging Visas with no work rights and no access to Medicare. The paper examines several key questions: What is the current state of play, in terms of health screening and medical care policies, for asylum seekers and refugees? Relatedly, how has current policy changed from that of the past? How does Australia compare with other countries in relation to health policy for asylum seekers and refugees? These questions are addressed with the aim of providing a clear description of the current situation concerning Australian health policy on access to medical care for asylum seekers and refugees. Issues concerning lack of access to appropriate health care and related services are raised, ethical and practical issues are explored, and current policy gaps are investigated.
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ItemA novel BH3 ligand that selectively targets Mcl-1 reveals that apoptosis can proceed without Mcl-1 degradation(ROCKEFELLER UNIV PRESS, 2008-01-28)Like Bcl-2, Mcl-1 is an important survival factor for many cancers, its expression contributing to chemoresistance and disease relapse. However, unlike other prosurvival Bcl-2-like proteins, Mcl-1 stability is acutely regulated. For example, the Bcl-2 homology 3 (BH3)-only protein Noxa, which preferentially binds to Mcl-1, also targets it for proteasomal degradation. In this paper, we describe the discovery and characterization of a novel BH3-like ligand derived from Bim, Bim(S)2A, which is highly selective for Mcl-1. Unlike Noxa, Bim(S)2A is unable to trigger Mcl-1 degradation, yet, like Noxa, Bim(S)2A promotes cell killing only when Bcl-x(L) is absent or neutralized. Furthermore, killing by endogenous Bim is not associated with Mcl-1 degradation. Thus, functional inactivation of Mcl-1 does not always require its elimination. Rather, it can be efficiently antagonized by a BH3-like ligand tightly engaging its binding groove, which is confirmed here with a structural study. Our data have important implications for the discovery of compounds that might kill cells whose survival depends on Mcl-1.
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ItemTWEAK-FN14 signaling induces lysosomal degradation of a cIAP1-TRAF2 complex to sensitize tumor cells to TNFα(ROCKEFELLER UNIV PRESS, 2008-07-14)Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor kappaB (NF-kappaB) signaling, and sensitize cells to tumor necrosis factor alpha (TNFalpha). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1-Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1-TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-kappaB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFalpha-induced death occurs. TWEAK-induced loss of the cIAP1-TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFalpha-induced death, whereas primary cells remain resistant. Conversely, cIAP1-TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFalpha sensitization. Lysosomal degradation of cIAP1-TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells.